Alleviation of noxious effects of cattle ranch composts on wheat seed germination by inoculation with Azospirillum spp.

Two commonly-used composts from dairy cow manure that are used to improve poor structure and fertility of desert soils have inhibitory effects on wheat seed germination, probably as a result of their high levels of humic acids. Inoculation of wheat seeds with two species of the plant growth-promoting bacteria Azospirillum brasilense Cd and A. lipoferum JA4 (separately) prior to sowing in these amended soils improved germination, similar to the natural level of germination of seeds in desert soil without compost amendment. Both compost amendments increased height of wheat seedlings in the range of 20–25%, increased shoot dry weight by 15–19%, but severely decreased (51–54% less) root dry weight. Inoculation of wheat seeds with A. brasilense Cd, but not with A. lipoferum JA4, significantly increased plant growth parameters (height, shoot and root dry weight) over control plants grown in soil-compost mixtures. This bacterial species could survive for a period of 20 days in compost humic acid solution, could increase its population when the humic acids served as the sole carbon source, and may change the composition of humic acids in which it grows. We suggest that inoculation with A. brasilense may alleviate noxious effects on germinating seeds caused by compost application by possibly transforming the composition of humic acids in the compost.     

Azospirillum — plant root associations: A review

Summary Bacteria of the genus Azospirillum are extensively studied for their plant-growth promoting effect following inoculation. Physiological and biochemical studies of these diazotrophic bacteria are now benefiting from recent breakthroughs in the development of genetic tools for Azospirilum. Moreover, the identification and cloning of Azospirillum genes involved in N₂ fixation, plant interaction, and phytohormone production have given new life to many research projects on Azospirillum. The finding that Azospirillum genes can complement specific mutations in other intensively studied rhizosphere bacteria like Rhizobia will certainly trigger the exploration of new areas in rhizosphere biology. Therefore a review of the Azospirillum-plant interactions is particularly timely.     

Inoculation of rice with nitrogen-fixing bacteria —problems and perspectives

Summary The present status and merits of inoculating rice with N₂-fixing bacteria are discussed in the light of recent advances. Bacterial inoculation improves plant growth and rice yield but not uniformly. The yield response to inoculation is more pronounced in the presence of moderate levels of fertilizer N. Evidence for the establishment and activity of the inoculated bacteria is limited, and the poor survival of the inoculum under field conditions further complicates the effects of inoculation. There is no clear evidence that improved growth and mineral content following inoculation are due to increased N₂ fixation. Beneficial effects of the inoculum on rice, such as plant growth promotion, N₂ fixation and antagonism effects against pathogens need to be further investigated under laboratory and field conditions. Improved management practices, such as organic amendments, suitable water and soil management, selection of efficient microbial strains, selection of effective breeding lines with high associative nitrogen fixation, and better management of agrochemicals are some of the measures suggested for deriving benefits from bacterial associations with rice.     

Increased emergence of spring wheat after inoculation with Pseudomonas chlororaphis isolate 2E3 under field and laboratory conditions

Inoculation at the time of planting with Pseudomonas chlororaphis strain 2E3 increased the emergence of spring wheat by 8% and 6% at two different sites in northern Utah. Isolate 2E3 strongly inhibited the growth of the wheat pathogen Fusarium culmorum on artificial media. A second isolate of P. chlororaphis (strain O6) also inhibited fungal growth on artifical media but did not increase emergence of the spring wheat at the same field sites. Inoculation of winter wheat by 2E3 did not promote emergence when planted into field soil sterilized by fumigation with methyl bromide. Under laboratory conditions, emergence of spring wheat in sterilized soils from both sites was at least 90%. In the soils that were not sterilized, emergence was below 25% in soil from one site and below 50% in soil from the other. Treating seeds with 2E3 significantly improved emergence in a sterile soil-containing matrix that had been inoculated with the wheat pathogen Fusarium culmorum. Consequently, we propose that increases in wheat emergence can be attributed to the suppression by 2E3 of pathogenic organisms present in the native field soils. A strain of Rhizoctonia solani, shown to the pathogenic on winter wheat, was isolated from one of these soils.     

Maize genotype effects on the response to Azospirillum inoculation

Two field experiments were carried out in Northern Argentina, during the 1989–1990 and 1990–1991 growing seasons, on Argentinian and Brazilian maize genotypes. The inoculant consisted of a mixture of four Azospirillum brasilense strain isolated from surface-sterilized maize roots in Argentina and three A. lipoferum strains isolated from surface-sterilized maize or sorghum roots in Brazil. Establishment of the inoculated strains was confirmed by the antibiotic resistance of the strains in the highest dilution vials. In all treatments, numbers of Azospirillum spp. were increased and the inoculated strains were found in the highest dilutions. While grain yields of the different genotypes varied between 1700 and 7300 kg ha^-1, total N accumulation was much less variable. Significant inoculation effects on total N accumulation and on grain yields were consistently negative with one Argentinian genotype and positive with four Argentinian and two Brazilian genotypes. Significant inoculation effects on leaf nitrate reductase activity at the flowering stage, observed in the range-55% to +176%, indicated the presence of various interactions between the plant NO _inf3 ^sup- metabolism and Azospirillum spp. Three Brazilian and one Argentinian maize genotype showed significant decreases in leaf nitrate reductase due to inoculation while four Argentinian genotypes showed significant increases in leaf nitrate reductase activity. The results of the present study, were consistent over the two field experiments and strongly indicate that more detailed plant genotype-Azospirillum spp. strain interaction studies, taking the entire N metabolism in the plant into account, are needed to allow better inoculation results of cereal crops.     

Seed inoculation with <Emphasis Type="Italic">Bacillus</Emphasis> spp. improves seedling vigour in oil-seed plant <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.

Jatropha (Jatropha curcas L.) is a non-edible oil-seed plant with adaptability to marginal semi-arid lands and wastelands. The Indian Government is promoting jatropha to reduce dependence on the crude oil and to achieve energy independence by the year 2012, under the National Biodiesel Mission. Selected strains of Bacillus spp., either supplemented with or without chitin, were tested for their ability to promote growth of jatropha seedlings in pot culture studies. The strains supported growth of jatropha seedlings up to 42 days after sowing. Among all strains, Bacillus pumilus (IM-3) supplemented with chitin showed over all plant growth promotion effect resulting in enhanced shoot length (113%), dry shoot mass (360%), dry root mass (467%), dry total plant mass (346%), leaf area (256%), and chlorophyll content (74%) over control. Treating seeds with strain IM-3 without chitin resulted in enhanced dry shoot mass (473%), dry total plant mass (407%), and chlorophyll content (82%). However, Bacillus polymyxa (KRU-22) with chitin supported maximum root length (143%). Either strain IM-3 alone or in combination with other promising strains could be promoted further for enhanced initial seedling growth of jatropha.     

Mitigation of salt stress in wheat seedlings by a <Emphasis Type="Italic">gfp</Emphasis>-tagged <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis>

Root colonization and mitigation of NaCl stress on wheat seedlings were studied by inoculating seeds with Azospirillum lipoferum JA4ngfp15 tagged with the green fluorescent protein gene (gfp). Colonization of wheat roots under 80 and 160 mM NaCl stress was similar to root colonization with this bacterial species under non-saline conditions, that is, single cells and small aggregates were mainly located in the root hair zone. These salt concentrations had significant inhibitory effects on development of seedlings, but not on growth in culture of gfp-A. lipoferum JA4ngfp15. Reduced plant growth (height and dry weight of leaves and roots) under continuous irrigation with 160 mM NaCl was ameliorated by bacterial inoculation with gfp-A. lipoferum JA4ngfp15. Inoculation of plants subjected to continuous irrigation with 80 mM NaCl or to a single application of either NaCl concentration (80 or 160 mM NaCl) did not mitigate salt stress. This study indicates that, under high NaCl concentration, inoculation with modified A. lipoferum reduced the deleterious effects of NaCl; colonization patterns on roots were unaffected and the genetic marker did not induce undesirable effects on the interaction between the bacterium and the plants.     

Influence of a plant growth-promoting pseudomonad and vesicular-arbuscular mycorrhizal fungus on alfalfa and birdsfoot trefoil growth and nodulation

Summary In a growth chamber study we examined the influence of a plant growth-promoting rhizobacterium, Pseudomonas putida R-20, and an acid-tolerant vesicular-arbuscular mycorrhizal (VAM) fungus, Glomus intraradices 25, on Medicago sativa L. and Lotus corniculatus L. growth and nodule development. Seedlings were planted in an acidic (pH 5.5), P-deficient soil containing re-established native microflora (minus VAM) and appropriate rhizobia, and inoculated with the rhizobacterium, the VAM fungus, or both. The plants were assayed at three intervals for up to 10–11 weeks. The growth-promoting rhizobacteria alone increased alfalfa shoot mass by 23% compared to all other treatments, but only at 8 weeks of growth, apparently by promoting nodulation and N₂ fixation (acetylene reduction activity). The presence of VAM, either alone or in combination with the rhizobacteria, generally decreased root length but only at 8 weeks also. As a group, the inoculation treatments increased all nodular measurements by 10 weeks of growth. Few treatment effects were found at 7 and 9 weeks for birdsfoot trefoil; neither plant nor nodular measurements differed among treatments. By 11 weeks, shoot mass was increased by the rhizobacteria alone by 36% compared to the control. As a group, the inoculation treatments all showed increased nodular responses by this time. The rhizobacteria stimulated mycorrhizal development on both plant species, but only at the initial samplings. No synergistic effects between the plant growth-promoting rhizobacterium and VAM inoculation were found. Although these results lend credence to the concept of managing microorganisms in the rhizosphere to improve plant growth, they emphasize the necessity for a more thorough understanding of microbial interactions as plants mature.     

Molecular characterization and PCR detection of a nitrogen-fixing Pseudomonas strain promoting rice growth

Nitrogen-fixing plant growth-promoting rhizobacteria (PGPR) from the genus Pseudomonas have received little attention so far. In the present study, a nitrogen-fixing phytohormone-producing bacterial isolate from kallar grass (strain K1) was identified as Pseudomonas sp. by rrs (16S ribosomal RNA gene) sequence analysis. rrs identity level was high with an uncharacterized marine bacterium (99%), Pseudomonas sp. PCP2 (98%), uncultured bacteria (98%), and Pseudomonas alcaligenes (97%). Partial nifH gene amplified from strain K1 showed 93% and 91% sequence similarities to those of Azotobacter chroococcum and Pseudomonas stutzeri, respectively. The effect of Pseudomonas strain K1 on rice varieties Super Basmati and Basmati 385 was compared with those of three non-Pseudomonas nitrogen-fixing PGPR (Azospirillum brasilense strain Wb3, Azospirillum lipoferum strain N4 and Zoogloea strain Ky1) used as single-strain inoculants. Pseudomonas sp. K1 was detected in the rhizosphere of inoculated plants by enrichment culture in nitrogen-free growth medium, which was followed by observation under the microscope as well as by PCR using a rrs-specific primer. For both rice varieties, an increase in shoot biomass and/or grain yield over that of noninoculated control plants was recorded in each inoculated treatment. The effect of Pseudomonas strain K1 on grain yield was comparable to those of A. brasilense Wb3 and Zoogloea sp. Ky1 for both rice varieties. These results show that nitrogen-fixing pseudomonads deserve attention as potential PGPR inoculants for rice.     

Biological nitrogen fixation in Azospirillum strain-maize genotype associations as evaluated by the 15N isotope dilution technique

Few studies of the inoculation of cereal crops with N₂-fixing bacteria have included more than one or two plant genotypes. In a recent study performed in Argentina using 12 different maize genotypes, it was found in 2 consecutive field experiments that several of them responded consistently, either negatively or positively, to inoculation with a mixture of strains of Azospirillum spp. The present study in post was performed to investigate the effect of inoculation of individual strains (and a mixture) of Azospirillum spp., and their nitrate reductase negative (NR-) mutants, on the growth of four of these maize genotypes. Two of these genotypes were grown in ¹⁵N-labelled soil with the aim of quantifying any contributions of biological N₂ fixation. Two genotypes (Morgan 318 and Dekalb 4D-70) produced similar increases in grain yield when they were inoculated with a mixture of Azospirillum spp. strains or fertilized with the equivalent of 100 kg N ha^-1. The other genotypes (Dekalb 2F-11 and CMS 22) showed little response to inoculation or N fertilization. The Morgan 318 and Dekalb 4D-70 genotypes showed a large increase in total N accumulation, suggesting that the response was due to increased N acquisition, but not due to bacterial nitrate reductase as the NR- mutants generally caused plant responses similar to those of the parent strains. Despite problems with the stabilization of the ¹⁵N enrichment in the soil, the ¹⁵N isotope dilution results indicated that there were very significant biological nitrogen fixation (BNF) contributions to the Dekalb 4D-70 and CMS 22 maize genotypes.Dedicated to Professor J.C.G. Ottow on the occasion of his 60th birthday     

Ozone effects on dry matter partitioning and chlorophyll fluorescence during plant development of wheat

In closed-chamber fumigation experiments dry matter partitioning and chlorophyll fluorescence of wheat were studied, analysing the effects of ozone during different stages of plant development. Ozone causes enhanced leaf senescence, leading to a loss of green leaf area and, consequently to a decreased supply of assimilates, affecting (in increasing order of severeness) stem, ear and grain productivity because of reduced storage pools for translocation. Leaves of plants before shooting stage were most sensitive but the lack of green leaf area after ear emergence had the most pronounced effects on grain yield.Measurements of photochemical capacity showed that evidence for negative ozone effects could be found in changes of chlorophyll fluorescence parameters in leaf sections not yet showing visible ozone injury. Negative effects on photosynthesis were more distinct with increasing accumulated ozone dose, with increasing age of leaf tissue and with increasing ozone sensitivity of the cultivar. The changes in chlorophyll fluorescence are most likely to be explained by a decreased pool size of plastoquinones caused by ozone.     

Increase in auxiliary photoprotective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense

Inoculation of wheat seedlings with the plant growth-promoting bacterium Azospirillum brasilense Cd was immobilized in alginate microbeads and, without applying any stress, significantly increased the quantity of several photosynthetic pigments, such as chlorophyll a, chlorophyll b, and the auxiliary photoprotective pigments violaxanthin, zeaxanthin, antheroxanthin, lutein, neoxanthin, and β-carotene. This resulted in greener plants with no apparent visible stress. After monitoring the quantity of photosynthetic pigments for 4 weeks, we observed that inoculated plants had higher quantities of pigments in shoot and stem. The greatest difference in the quantity of all pigments between inoculated and noninoculated plants occurred in the first week of growth. Regardless of treatment, the quantity of pigments in stems was three to four times less than the quantity of these pigments in shoots. Application of Azospirillum, either as liquid inoculant or as alginate microbeads, did not alter the positive effect of the bacteria on pigment production or the positive response of the plants towards A. brasilense Cd inoculation.     

Dual inoculation of pretransplant stage Oryza sativa L. plants with indigenous vesicular-arbuscular mycorrhizal fungi and fluorescent Pseudomonas spp.

Summary This study examined the response of rice (Oryza sativa L.) plants at the pretransplant/nursery stage to inoculation with vesicular-arbuscular mycorrhizal (VAM) fungi and fluorescent Pseudomonas spp., singly or in combination. The VAM fungi and fluorescent Pseudomonas spp. were isolated from the rhizosphere of rice plants. In the plants grown in soil inoculated with fluorescent Pseudomonas spp. alone, I found increases in shoot growth, and in root length and fine roots, and decreases in root growth, and P and N concentrations. In contrast, in the plants colonized by VAM fungi alone, the results were the reverse of those of the pseudomonad treatment. Dual inoculation of soil with VAM fungi and fluorescent Pseudomonas spp. yielded plants with the highest biomass and nutrient acquisition. In contrast, the plants of the control treatment had the lowest biomass and nutrient levels. The dual-inoculated plants had intermediate root and specific root lengths. The precentages of mycorrhizal colonization and colonized root lengths were significantly lower in the dual-inoculated treatment than the VAM fungal treatment. Inoculation of plants with fluorescent Pseudomonas spp. suppressed VAM fungal colonization and apparently reduced photosynthate loss to the mycorrhizal associates, which led to greater biomass and nutrient levels in dual-inoculated plants compared with plants inoculated with VAM fungi alone. Dual inoculation of seedlings with fluorescent Pseudomonas spp. and VAM fungi may be preferable to inoculation with VAM alone and may contribute to the successful establishment of these plants in the field.     

Responses of native legume desert trees used for reforestation in the Sonoran Desert to plant growth-promoting microorganisms in screen house

Three slow-growing legume trees used for desert reforestation and urban gardening in the Sonoran Desert of Northwestern Mexico and the Southwestern USA were evaluated whether their growth can be promoted by inoculation with plant growth-promoting bacteria (Azospirillum brasilense and Bacillus pumilus), unidentified arbuscular mycorrhizal (AM) fungi (mainly Glomus sp.), and supplementation with common compost under regular screenhouse cultivation common to these trees in nurseries. Mesquite amargo (Prosopis articulata) and yellow palo verde (Parkinsonia microphylla) had different positive responses to several of the parameters tested while blue palo verde (Parkinsonia florida) did not respond. Survival of all tree species was over 80% and survival of mesquite was almost 100% after 10 months of cultivation. Inoculation with growth-promoting microorganisms induced significant effects on the leaf gas exchange of these trees, measured as transpiration and diffusive resistance, when these trees were cultivated without water restrictions.     

Potential of Trichoderma spp. as consistent plant growth stimulators

In a series of repeated trials, six Trichoderma spp. strains, applied as a dried powder from a liquid fermentation in molasses/yeast medium, proved to be consistent at promoting the growth of lettuce (Latuca sativa L.) seedlings grown in a peat-sand potting compost in the glasshouse. Strains WT, 92, 20, and 75 at 0.75% or 1% w:w concentrations increased shoot dry weight by up to 26%, although WT did inhibit germination. For example, after 4 days only 13% of seeds sown in WT 1% w:w treated compost had germinated, whereas in other treatments germination was consistently greater than 32%. WT increased shoot fresh and dry weights by 14.3 g and 0.6 g per pot, respectively, without affecting the root dry weights, to give concomitant increases in shoot: root ratios of fresh and dry weight. The potential use of these Trichoderma spp. strains for plant growth promotion is discussed.     

Nitrogen-fixing potential of Acacia mangium and Acacia auriculiformis seedlings inoculated with Bradyrhizobium and Rhizobium spp.

Summary Two Australian Acacia species, A. mangium and A. auriculiformis were inoculated in vitro with eight strains of Bradyrhizobium spp. and two strains of Rhizobium spp. On the two plant species, only Bradyrhizobium spp. strains formed effective N₂-fixing nodules. A. mangium, which nodulates effectively with a restricted range of Bradyrhizobium spp. strains, is a specific host compared to A. auriculiformis. A. auriculiformis is assumed to be a promiscuous host because it nodulates effectively with a wide range of Bradyrhizobium spp. strains. Nodule efficiency as expressed by the ratio of N₂ fixed to nodule dry weight appeared to be higher in A. auriculiformis (0.44–0.81) than in A. mangium (0.23–0.55).     

Influence of host seed on metabolic activity of Enterobacter cloacae in the spermosphere

Little is known regarding the influences of nutrients released from plants on the metabolic activity of colonizing microbes. To gain a better understanding of these influences, we used bioluminescence- and oxygen consumption-based methods to compare bacterial metabolic activity expressed during colonization of two different seed types. Metabolic activity expressed by Enterobacter cloacae during colonization of pea seeds, which exude high levels of reduced carbon nutrients, was compared with that during colonization of cucumber seeds, which exude orders of magnitude less reduced carbon nutrients. Metabolic activity levels expressed by E. cloacae populations were much higher throughout a 72 h colonization period on pea seed compared with those observed on cucumber seed, directly correlating metabolic activity level with amounts of nutrients released by seeds. In vitro studies indicated E. cloacae cells expressed different levels of metabolic activity when incubated with different individual carbohydrates commonly found in cucumber and pea seed exudates. The addition of exogenous carbohydrate to cucumber seed increased metabolic activity expressed by colonizing E. cloacae; with the level of increase dependent on both quantity and type of carbohydrate supplement. Subtraction of carbohydrate available to E. cloacae on cucumber and pea seeds through mutation in pfkA decreased metabolic activity by this bacterium. Results presented here provide strong evidence that metabolic activity of spermosphere-colonizing bacteria is host dependent, and that levels of activity are based largely on both quantitative and qualitative composition of host exudate released during seed germination.     

Co-inoculation of selected nodule endophytic rhizobacterial strains with Rhizobium tropici promotes plant growth and controls damping off in common bean

Production of common bean(Phaseolus vulgaris)is limited by the occurrence of damping off(rhizoctoniosis),which is caused by the fungus Rhizoctonia solani.However,the co-inoculation of plant growth-promoting rhizobacteria(PGPR)involved in biological control along with diatomic nitrogen(N2)-fixing rhizobia can enhance N nutrition and increase production.In this context,finding microorganisms with synergistic effects that perform these two roles is of fundamental importance to ensure adequate yield levels.The aim of this study was to evaluate the effects of co-inoculation of nodule endophytic strains of the genera Bacillus,Paenibacillus,Burkholderia,and Pseudomonas with Rhizobium tropici CIAT 899,an N2-fixing rhizobial strain,on the biocontrol of damping off and growth promotion in common bean plants.Greenhouse experiments were conducted under axenic conditions using the common bean cultivar Pérola.The first experiment evaluated the potential of the 14 rhizobacterial strains,which were inoculated alone or in combination with CIAT 899,for the control of R.solani.The second experiment evaluated the ability of these 14 rhizobacterial strains to promote plant growth with three manners of N supply:co-inoculation with CIAT 899 at low mineral N supply(5.25 mg N mL^-1),low mineral N supply(5.25 mg N mL^-1),and high mineral N supply(52.5 mg N mL^-1).The use of rhizobacteria combined with rhizobia contributed in a synergistic manner to the promotion of growth and the control of damping off in the common bean.Co-inoculation of the strains UFLA 02-281/03-18(Pseudomonas sp.),UFLA 02-286(Bacillus sp.),and UFLA 04-227(Burkholderia fungorum)together with CIAT 899 effectively controlled damping off.For the common bean,mineral N supply can be replaced by the co-inoculation of CIAT 899 with plant growth-promoting strains UFLA 02-281/02-286/02-290/02-293.Nodule endophytes UFLA02-281/02-286 are promising for co-inoculation with CIAT 899 in the common bean,promoting synergy with rhizobial inoculation and protection against disease.     

Ecology of soil microflora and mycorrhizal symbionts in degraded forests at two altitudes

Summary Microbial populations were estimated in four different forest stands at different regenerational stages, two each at higher and lower altitudes. The fungal and bacterial populations showed marked seasonal variations at both altitudes. Quantitatively, the bacterial population was higher than the fungal population. Although 25 fungal species were isolated at the lower altitude, only 15 were obtained at the higher altitude. Penicillium chrysogenum and Trichoderma viride were dominant at the lower and higher altitudes, respectively. In the more degraded forest stand at the lower altitude both the fungal and the bacterial population showed a significant positive correlation with organic C (r=0.658 and 0.735, respectively), whereas in the less degraded forest stand there was a significant correlation only between the fungal population and organic C (r=0.835). At the higher altitude, however, a highly significant correlation (P<0.05) was observed between the fungal population, soil moisture and organic C in both the forest stands. Disturbance to the soil and vegetation adversely affected the microbial population, and also affected endogonaceous spores. At the lower altitude, plants in the more degraded forest stand were more mycotrophic compared to those in the less degraded stand. The level of mycorrhizal infection showed a highly positive correlation with soil moisture, organic C, total N, and available P. The spore population, however, was correlated negatively with these parameters. Three different endogonaceous genera, Glomus, Gigaspora, and Acaulospora, were identified during the course of investigation. Glomus, however, was dominant.